University of Guelph View Institution's Website 19 articles published in JoVE Biology Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks Davier Gutierrez-Gongora1, Fouad Raouf-Alkadhimi1, Ryan S. Prosser2, Jennifer Geddes-McAlister1 1Molecular and Cellular Biology Department, University of Guelph, 2School of Environmental Sciences, University of Guelph The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production. Cancer Research Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid Jacob G. E. Yates1, Alexander Leacy1, Phuc H. Pham1, Nicole Zielinska1, Emily A. Tusnadi1, Leonardo Susta1, Sarah K. Wootton1 1Department of Pathobiology, University of Guelph Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields > 6 × 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described. Behavior Assessment of Mouse Judgment Bias through an Olfactory Digging Task Aileen MacLellan*1, Agustina Resasco*2,3, Lauren Young4, Georgia Mason1 1Department of Integrative Biology, University of Guelph, 2Institute of Cell Biology and Neurosciences, National Scientific and Technical Research Council-University of Buenos Aires, 3Laboratory of Experimental Animals, Faculty of Veterinary Sciences, National University of La Plata, 4Department of Animal Biosciences, University of Guelph This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed. Neuroscience Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat Diana J. Wallin*1,2, Emily D. K. Sullivan*1,2, Elise M. Bragg1, Jibran Y. Khokhar2,4, Hanbing Lu2,3, Wilder T. Doucette1,2 1Dartmouth-Hitchcock Medical Center, 2Geisel School of Medicine at Dartmouth, 3National Institute on Drug Abuse, National Institutes of Health, 4University of Guelph This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine. Biochemistry Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases Olivia Roscow1, Wei Zhang1 1Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination. Immunology and Infection Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models Amira D. Rghei1, Brenna A. Y. Stevens*1, Sylvia P. Thomas*1, Jacob G. E. Yates*1, Benjamin M. McLeod1, Khalil Karimi1, Leonardo Susta1, Byram W. Bridle1, Sarah K. Wootton1 1Department of Pathobiology, University of Guelph Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields >1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies. Environment Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils. Reza Khalidy*1, Fatima Haque*1, Yi Wai Chiang1, Rafael M. Santos1 1School of Engineering, University of Guelph The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields. Immunology and Infection Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions Brianna Ball1, Arjun Sukumaran1, Jennifer Geddes-McAlister1 1Molecular and Cellular Biology Department, University of Guelph Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment. Biochemistry Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics Erin M. Anderson1, Neil A. Greenwood1, Dyanne Brewer2, Cezar M. Khursigara1 1Department of Molecular and Cellular Biology, University of Guelph, 2Mass Spectrometry Facility, University of Guelph This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software. Biology Field Identification of Matricaria chamomilla using a Portable qPCR System Zhengxiu Yang1, Zheng Quan2, Tiffany Chua3, Leo Li1, Yanjun Zhang3, Silva Babajanian2, Francesco Buongiorno4, Isabella Della Noce4, Lorenzo Colombo4, Steven Newmaster5, Tricia Chua3, Peter Chang3, Gary Swanson3, Zhengfei Lu2 1Herbalife NatSource (Hunan) Natural Products Co., Ltd., 2Corporate Quality Laboratory, Herbalife International of America, Inc., 3Corporate Quality, Herbalife International of America, Inc., 4Hyris Ltd, 5Natural Health Product Research Alliance, University of Guelph Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses. Immunology and Infection Antibiotic Dereplication Using the Antibiotic Resistance Platform Haley L. Zubyk1, Georgina Cox2, Gerard D. Wright1 1Department of Biochemistry and Biomedical Sciences, M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 2Department of Molecular and Cellular Biology, University of Guelph We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient. Biology Bovine Mammary Gland Biopsy Techniques Veridiana L. Daley1,4, Charles Dye2, Sophie H. Bogers3, R. Michael Akers4, Francisco C. Rodriguez3, John P. Cant5, John Doelman5, Peter Yoder4, Karan Kumar2, Dane Webster6, Mark D. Hanigan4 1National Animal Nutrition Program, a National Research Support Project (NRSP-9), Department of Animal and Food Sciences, University of Kentucky, 2School of Performing Arts, Virginia Tech, 3Department of Large Animal Clinical Sciences, The Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 4Department of Dairy Science, Virginia Tech, 5Department of Animal Biosciences, University of Guelph, 6School of Visual Arts, Virginia Tech This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations. Neuroscience Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method Emma L. Louth1, Charles D. Sutton1, Ari L. Mendell1, Neil J. MacLusky1, Craig D.C. Bailey1 1Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage. Engineering Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent Evangelos Georgakopoulos1, Rafael M. Santos2, Yi Wai Chiang3, Vasilije Manovic4 1Department of Offshore, Process and Energy Engineering, Cranfield University, 2School of Applied Chemical and Environmental Sciences, Sheridan College Institute of Technology and Advanced Learning, 3School of Engineering, University of Guelph, 4Carbon Systems Engineering, Centre for Combustion, Carbon Capture and Storage, Cranfield University A protocol for the parallel production of precipitated calcium carbonate and zeolitic material from blast furnace slag via mineral carbonation and alkaline hydrothermal conversion, respectively, is presented. The performance of the zeolitic material towards nickel adsorption is tested. Genetics Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions Sara Timpano*1, Gaelan Melanson*1, Sonia L. Evagelou1, Brianna D. Guild1, Erin J. Specker1, James Uniacke1 1Department of Molecular and Cellular Biology, University of Guelph Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners. Bioengineering Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy Eric Birkenhauer1, Suresh Neethirajan1 1BioNano Laboratory, School of Engineering, University of Guelph Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces. Behavior A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration AnneMarie Levy1, Cheryl L. Limebeer1, Justin Ferdinand1, Ucal Shillingford1, Linda A. Parker1, Francesco Leri1 1Department of Psychology, University of Guelph The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e. lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement. Neuroscience Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization Graham S.T. Smith1, Janine A.M. Voyer-Grant1, George Harauz1 1Department of Molecular and Cellular Biology, University of Guelph Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues. Biology Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts Steve P. Hawley*1, Melanie K. B. Wills*1, Nina Jones1 1Department of Molecular and Cellular Biology, University of Guelph In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.